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MicroRNA regulation of human protease genes essential for influenza virus replication.

Meliopoulos VA, Andersen LE, Brooks P, Yan X, Bakre A, Coleman JK, Tompkins SM, Tripp RA - PLoS ONE (2012)

Bottom Line: However, the rapid emergence of drug resistance has emphasized the need for new drug targets.The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis.Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

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RNAi of 5 host protease genes down-regulated influenza virus replication.A: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cytotoxicity was determined by adenylate kinase (AK) release. Cells treated with the siTOX control were considered 100% cytotoxic and all values were normalized to siTOX. RLU = relative luciferase units. * p<0.05 vs siNEG, ** p<0.01 vs siNEG, ***p<0.005 vs siNEG; siTOX vs all samples: p<0.001 (not shown). Line shows 20% of siTOX control. B: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. 48 hours post-infection, cells were fixed in 4% formaldehyde and stained with an anti-NP (green) monoclonal antibody followed by counterstain with DAPI (blue.) Positive (+) control: siMEK, negative (−) control: siNEG. Magnification is 20× (bar is 100 microns). C: Cells were transfected with 100 nM of a novel siRNA targeting a different seed site from the SMARTpool used in the primary screen and infected as in B. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs siNEG).
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pone-0037169-g001: RNAi of 5 host protease genes down-regulated influenza virus replication.A: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cytotoxicity was determined by adenylate kinase (AK) release. Cells treated with the siTOX control were considered 100% cytotoxic and all values were normalized to siTOX. RLU = relative luciferase units. * p<0.05 vs siNEG, ** p<0.01 vs siNEG, ***p<0.005 vs siNEG; siTOX vs all samples: p<0.001 (not shown). Line shows 20% of siTOX control. B: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. 48 hours post-infection, cells were fixed in 4% formaldehyde and stained with an anti-NP (green) monoclonal antibody followed by counterstain with DAPI (blue.) Positive (+) control: siMEK, negative (−) control: siNEG. Magnification is 20× (bar is 100 microns). C: Cells were transfected with 100 nM of a novel siRNA targeting a different seed site from the SMARTpool used in the primary screen and infected as in B. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs siNEG).

Mentions: A primary RNAi screen of 481 host protease genes in a human type II respiratory epithelial cell line (A549) identified 24 genes important for A/WSN/33 influenza virus replication (Figure S1). The gene hits identified were ≥1.5σ and ≤−1.5σ from the mean Z-score. Endpoint validation of the gene hits in the primary screen included influenza nucleoprotein (NP) cell localization determined by immunohistochemistry, determining the level of infectious virus by TCID50 assay, as well as influenza matrix (M) gene copy number determined by qPCR (data not shown). For validation of primary gene hits, a novel siRNA targeting the same gene but at a different seed site (Table 1) was required to produce the same phenotype as observed in the screen. Protein levels of the protease genes were assessed to ensure that RNAi silencing was decreasing target gene expression (Figure S2). After validation, five genes were identified that decreased virus replication when silenced: ADAMTS7, CPE, DPP3, MST1, and PRSS12 (Table 1). Silencing of these protease genes did not cause cytotoxicity in A549 cells compared to the cytotoxicity (siTOX) control. siMEK (an siRNA targeting MAP2K) and siNEG (a negative, non-targeting siRNA control) both displayed low level cytotoxicity possibly due to the importance of MEK in cellular signaling, and the induction of an inflammatory response to siNEG due to off-target effects (Figure 1A). Silencing of the five validated genes resulted in considerably decreased influenza NP staining compared to the negative (−) control, and was consistent with low NP staining in cells treated with the positive (+) siRNA control, siMEK (Figure 1B). The level of infectious virus from A549 cells treated with 100 nM of a single siRNA targeting each of the validated genes was greatly (siCPE, siMST1 and siPRSS12) and significantly (p<0.05; siADAMTS7 and siDPP3) reduced (Figure 1C). Influenza virus M gene levels reflected the low level of infectious virus and NP staining observed for A549 cells treated with siRNAs targeting the five validated genes, as well (data not shown).


MicroRNA regulation of human protease genes essential for influenza virus replication.

Meliopoulos VA, Andersen LE, Brooks P, Yan X, Bakre A, Coleman JK, Tompkins SM, Tripp RA - PLoS ONE (2012)

RNAi of 5 host protease genes down-regulated influenza virus replication.A: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cytotoxicity was determined by adenylate kinase (AK) release. Cells treated with the siTOX control were considered 100% cytotoxic and all values were normalized to siTOX. RLU = relative luciferase units. * p<0.05 vs siNEG, ** p<0.01 vs siNEG, ***p<0.005 vs siNEG; siTOX vs all samples: p<0.001 (not shown). Line shows 20% of siTOX control. B: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. 48 hours post-infection, cells were fixed in 4% formaldehyde and stained with an anti-NP (green) monoclonal antibody followed by counterstain with DAPI (blue.) Positive (+) control: siMEK, negative (−) control: siNEG. Magnification is 20× (bar is 100 microns). C: Cells were transfected with 100 nM of a novel siRNA targeting a different seed site from the SMARTpool used in the primary screen and infected as in B. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs siNEG).
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Related In: Results  -  Collection

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pone-0037169-g001: RNAi of 5 host protease genes down-regulated influenza virus replication.A: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cytotoxicity was determined by adenylate kinase (AK) release. Cells treated with the siTOX control were considered 100% cytotoxic and all values were normalized to siTOX. RLU = relative luciferase units. * p<0.05 vs siNEG, ** p<0.01 vs siNEG, ***p<0.005 vs siNEG; siTOX vs all samples: p<0.001 (not shown). Line shows 20% of siTOX control. B: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. 48 hours post-infection, cells were fixed in 4% formaldehyde and stained with an anti-NP (green) monoclonal antibody followed by counterstain with DAPI (blue.) Positive (+) control: siMEK, negative (−) control: siNEG. Magnification is 20× (bar is 100 microns). C: Cells were transfected with 100 nM of a novel siRNA targeting a different seed site from the SMARTpool used in the primary screen and infected as in B. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs siNEG).
Mentions: A primary RNAi screen of 481 host protease genes in a human type II respiratory epithelial cell line (A549) identified 24 genes important for A/WSN/33 influenza virus replication (Figure S1). The gene hits identified were ≥1.5σ and ≤−1.5σ from the mean Z-score. Endpoint validation of the gene hits in the primary screen included influenza nucleoprotein (NP) cell localization determined by immunohistochemistry, determining the level of infectious virus by TCID50 assay, as well as influenza matrix (M) gene copy number determined by qPCR (data not shown). For validation of primary gene hits, a novel siRNA targeting the same gene but at a different seed site (Table 1) was required to produce the same phenotype as observed in the screen. Protein levels of the protease genes were assessed to ensure that RNAi silencing was decreasing target gene expression (Figure S2). After validation, five genes were identified that decreased virus replication when silenced: ADAMTS7, CPE, DPP3, MST1, and PRSS12 (Table 1). Silencing of these protease genes did not cause cytotoxicity in A549 cells compared to the cytotoxicity (siTOX) control. siMEK (an siRNA targeting MAP2K) and siNEG (a negative, non-targeting siRNA control) both displayed low level cytotoxicity possibly due to the importance of MEK in cellular signaling, and the induction of an inflammatory response to siNEG due to off-target effects (Figure 1A). Silencing of the five validated genes resulted in considerably decreased influenza NP staining compared to the negative (−) control, and was consistent with low NP staining in cells treated with the positive (+) siRNA control, siMEK (Figure 1B). The level of infectious virus from A549 cells treated with 100 nM of a single siRNA targeting each of the validated genes was greatly (siCPE, siMST1 and siPRSS12) and significantly (p<0.05; siADAMTS7 and siDPP3) reduced (Figure 1C). Influenza virus M gene levels reflected the low level of infectious virus and NP staining observed for A549 cells treated with siRNAs targeting the five validated genes, as well (data not shown).

Bottom Line: However, the rapid emergence of drug resistance has emphasized the need for new drug targets.The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis.Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

Show MeSH
Related in: MedlinePlus